Focal adhesion kinase (FAK) is a ubiquitously expressed signaling protein that links extracellular matrix (ECM)adhesion with activation of intracellular signaling pathways and gene expression. Human mesenchymal stem cells (hMSC) are a source of osteoblast precursors potentially suitable for use in tissue engineering applications to repair damaged bone. Controlled induction and maintenance of osteoblast differentiation is a significant hurdle in the quest to engineer bone. The role of FAK signaling in controlling osteogenic gene expression in hMSC is entirely unknown. Our long range objective is to devise a strategy for transplanting hMSC into sites of skeletal injury under conditions that maximally favor osteogenesis. The goal of this project is to define the role of FAK signaling during hMSC osteogenic differentiation and to establish optimal conditions for this differentiation. Our hypothesis is that FAK is a key signal transducing element in the pathway linking ECM binding and applied tensile strain to expression of osteoblast-specific genes and matrix mineralization in hMSC. Aims: (1) Quantify FAK phosphorylation/activation states, osteogenic gene expression levels, and matrix mineralization; in cultured hMSC cultured on purified ECM, and develop predictive modelslinking these parameters. (2) Assess the role of FAK phosphorylation in controlling osteogenic gene expression and matrix mineralization using mutant FAK constructs lacking tyrosine residues (Y397, Y576, Y925) important in FAK signaling, and (3) Quantify the effect of tensile strain on FAK phosphorylation, osteogenic gene expression, and matrix mineralization on cells plated on purified ECM proteins. Our rationale is that, once we establish a mechanistic understanding of FAK signaling pathways during hMSC osteogenesis, it may be possible to control hMSC osteogenesis by controlling FAK activity directly. We expect two significant outcomes: (i) quantitative models linking specific extracellular stimuli with FAK activity, osteogenic gene expression, and matrix mineralization; and (ii) definition of optimal extracellular stimuli that will control FAK activity and, ultimately, osteogenic differentiation of hMSC.